Stem cells are defined as cells that are capable of a differentiation into many other differentiated cell types. Embryonic stem cells are stem cells from embryos which are capable of differentiation into most, if not all, of the differentiated cell types of a mature body. Stem cells are referred to as pluripotent, which describes this capability of differentiating into many cell types. A category of pluripotent stem cell of high interest to the research community is the human embryonic stem cell, abbreviated here as hES cell, which is an embryonic stem cell derived from a human embryonic source. Human embryonic stem cells are of great scientific interest because they are capable of indefinite proliferation in culture and are thus capable, at least in principle, of supplying cells and tissues for replacement of failing or defective human tissue. The existence in culture of human embryonic stem cells offers the potential of unlimited amounts of human cells and tissues for use in a variety of therapeutic protocols to assist in human health. It is envisioned in the future human embryonic stem cells will be proliferated and directed to differentiate into specific lineages so as to develop differentiated cells or tissues which can be transplanted into human bodies for therapeutic purposes.
One of most significant features of human embryonic stem cells is the attribute of being capable of self-renewal. By that, it is meant that the hES cells are capable of proliferating into multiple progeny stem cells, each of which seems to have the full potential of its ancestor cell. In other words, the progeny are renewed to have all the developmental and proliferative capacity of the parental cell. This attribute, combined with the pluripotency, are the traits that make hES cells candidates for many potential uses, since, the theory, hES cells can be reproduced indefinitely and in large numbers and then induced to become any cell type in the human body. The attribute of ability to self-renew appears closely linked to the attribute of being undifferentiated in the sense that at least given present knowledge, only undifferentiated hES cells are capable of indefinite self-renewal and as soon as the cells differentiate, the attribute of self-renewal capability is lost. Since human embryonic stem cells will spontaneously differentiate, care must be taken in culture conditions to maintain the cells in an undifferentiated state.
Basic techniques to create and culture human embryonic stem cells to maintain the cells in an undifferentiated state have been described. The existing techniques do work, but there are limitations and drawbacks to some of the procedures currently used to culture human embryonic stem cells. One limitation is of particular concern. Most existing human embryonic stem cell lines have been, to one degree or another, exposed directly to mouse cells or to a medium in which mouse cells have been cultured previously. The original techniques for the generation and culture of human embryonic stem cells described the use of mouse embryonic fibroblast (MEF) feeder cells as a feeder layer on which human embryonic stem cells could be cultured. The fibroblast feeder layer acts, through some as yet incompletely understood mechanism, to enable the stem cells to remain in an undifferentiated state. Later, it was discovered that the same phenomenon could be achieved if the stem cells were exposed to “conditioned media.” A conditioned medium is nothing more than the stem cell culture medium which had previously been cultured on feeder cells such as MEF. Either the feeder cells impart some factor to the medium or remove some factor from the medium, but the result is that the conditioned medium can be used to culture stem cells without differentiation. Either culture condition, the direct growth on feeder cells, or the use of condition media, raises the concern that one or more agents such as a virus could transmit from the mouse cells to the human ES cells. If one of the objectives of human embryonic stem cell cultures is to create tissues which can ultimately be transplanted into a human body, it is highly desirable that the stem cells never have been exposed to cells of another species or to media which have been used to culture cells of another species. Also, the need for feeder cells of any species represents an unneeded biological variable in the culture of stem cells that is best avoided if possible. Accordingly, defining a culture condition, which will permit the proliferation and culture of human embryonic stem cells without a fibroblast feeder layer and without conditioned medium, is of great interest in the continued development of techniques for the use of human embryonic stem cells.
Several medium formulations will permit human ES cells to remain undifferentiated for some time, but that state often fails to maintain itself over long term culture. In particular, we define a passage as the growth of human ES cells from an initial seed culture in a culture plate to growth to cell confluence in the same culture plate as a “passage.” We have found several medium formulations that permit the cultivation of human ES cells for one or two passages without severe differentiation, but then the cells differentiate gradually or rapidly upon subsequent passages. We have come to believe that in order for a medium to truly support the indefinite proliferation of human ES cells without differentiation, without feeder cells or feeder conditioned medium, the medium must be demonstrated to support culture of human ES cells in a substantially uniform and undifferentiated state for at least five passages.